The original suggestion that thyrotropin-releasing hormon (TRH) was formed biosynthetically by a non-ribosomal pathway has been questioned by several laboratories. Results obtained in this laboratory suggest that a macromolecule present in frog brains can be converted to a "TRH-like material" which has physical, immunological and biological properties similar to the authentic hormone. This proposal describes the procedures which will be used to characterize and isolate the macromolecule which gives rise to the "TRH-like material." Upon isolation, its amino acid sequence will be determined. In addition, the mechanism of hormone amidation will be investigated. TRH degradation in brain and serum has been investigated by this laboratory. A homogeneous TRH deamidase has been purified from rat brain by the principal investigator. The physiochemical and kinetic properties of the enzyme will be determined. Efforts to isolate the serum TRH peptidase which cleaves the His-Pro bond of the hormone will utilize pGlu.His.beta napthylamide as a substrate analogue of TRH.